Novel dna polymerase

ABSTRACT

Provided are various novel DNA polymerases. 
     Provided are: a DNA polymerase comprising: an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 12, which has a substitution of arginine at position 651 by an amino acid residue having a negatively charged side chain, preferably by asparatic acid or glutamic acid, more preferably by glutamic acid; and a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 14, which has a substitution of proline at position 653 by an amino acid residue having a negatively charged side chain, preferably by asparatic acid or glutamic acid, more preferably by glutamic acid.

TECHNICAL FIELD

The present invention relates to novel DNA polymerases. The DNApolymerases of this invention are particularly useful for PCR.

BACKGROUND ART

DNA polymerases are enzymes that can synthesize new DNA strands alongtemplate DNA strands in vitro. DNA polymerases can synthesize new DNAstrands from a template DNA, an oligonucleotide serving as a primer, andfour types of deoxynucleotides (dATP, dGTP, dCTP, and dTTP). DNApolymerases are also used in many genetic engineering techniques,including nucleotide sequencing and PCR.

Thermostability of polymerases is essential for PCR, and the currentprotocol of nucleotide sequencing generally uses the cycle sequencingmethod using a thermostable DNA polymerase as a standard technique. Inorder to find a thermostable enzyme, one would usually search enzymesproduced by thermophilic microorganisms. Among thermophilic bacteria,those which proliferate at an optimum growth temperature of at least 80°C. are particularly referred to as “hyperthermophilic bacteria” andserve as excellent resources for thermostable enzymes. Taq DNApolymerases (also referred to as “Taq polymerases”) which are currentlywidely used in PCR were originally isolated from the thermophiliceubacterium Thermus aquaticus.

Based on the similarity in amino acid sequences, DNA polymerases arecategorized into seven groups: Families A, B, C, D, E, X and Y. Enzymesbelonging to the same family basically exhibit very similar properties.The enzymes that are in practical use are those belonging to Families Aand B.

Family A enzymes have superior performance in recognizingdideoxynucleotides as substrates and are most appropriate for nucleotidesequencing. Thus, the enzymes contained in currently commerciallyavailable sequencing kits are all those which belong to Family A and arederived from thermophilic eubacteria. In PCR, Family A and B enzymes areselectively used depending on the purpose.

Family B enzymes are not suitable for nucleotide sequencing because ofpoor incorporation of dideoxynucleotides but have 3′-5′ exonucleaseactivity which is involved in the accuracy in synthesizing DNA strandsaccording to the sequences of template strands—during amplification, theenzymes of this family produce less errors than Family A enzymes such asTaq polymerases with no exonuclease activity. The Family B enzymes thatare commercialized are those derived from hyperthermophilic archaea. Inorder to perform PCR more accurately, it is advisable to use Family Benzymes, whereas in order to amplify long-chain DNA, Family A enzymescan be selected due to their superior extensibility and superior DNAsynthesis efficiency.

Comparison between the two DNA polymerases that are derived frombacteria belonging to the genus Thermus and which have been up to nowwidely used as PCR enzymes shows that Taq DNA polymerase only has weakreverse transcriptional activity, while Tth DNA polymerase (“Tthpolymerase”) derived from Thermus thermophilus has significantly strongreverse transcriptional activity. This property of Tth polymerase isutilized in a simple RT-PCR technology in which a single enzyme is usedin a single reaction tube to synthesize cDNA from mRNA by reversetranscription and then amplify the synthesized cDNA. Since the optimumtemperature of this enzyme is high, the enzyme makes it possible toperform a reverse transcription reaction at relatively high temperatures(around 60° C.) and is also effective for the reverse transcription ofRNA which easily forms a three-dimensional structure, but the enzyme isnot suitable for the synthesis of long cDNAs like those reaching as longas several kilo bases in length.

PCR is a gene analysis technology that is widely used throughout theworld as a routinely utilized technique. Accordingly, there is a needfor a DNA polymerase that is more convenient, easier-to-use, and morereliable, and it is also desired to provide various DNA polymerases thatcan amplify various DNAs appropriately depending on the template to beused as well as the purpose to be needed for PCR such as extensibility,rapidity, and accuracy.

As regards modification of Taq polymerases, there have been hithertoreports stating that primers were designed based on the segments of anamino acid sequence highly conserved in Family A DNA polymerases, eachof which contains an active site, gene fragments were amplified by PCRusing DNA samples derived from hot spring soil as templates, and thecorresponding segments of a wild-type Taq polymerase gene weresubstituted by the amplified fragments, whereby obtained were chimericDNA polymerases with higher extension activity than Taq polymerases(Patent Documents 1 and 2). Another report showed that on the basis ofmetagenomic analysis and the three-dimensional structure information ofDNA polymerases, one or more mutations were introduced that produce anincreased the total electric charges of glutamic acid at position 742and alanine at position 743 in an amino acid sequence of a Taqpolymerase, whereby obtained was a modified Taq polymerase that issuperior to the Taq polymerase in at least one of: primer extensionactivity; binding activity on a primer annealed to a template DNA; andPCR performance (Patent Document 3).

CITATION LIST Patent Documents

Patent Document 1: Japanese Patent Application Publication No. JP2006-101791

Patent Document 2: Japanese Patent Application Publication No. JP2006-204267

Patent Document 3: Japanese Patent No. JP 4193997

SUMMARY OF INVENTION Technical Problem

The present inventors made detailed comparison between the amino acidsequences of Taq polymerases and Tth polymerases, focusing on thedifference in the properties associated with their amino acid sequenceidentity (about 80%) and reverse transcriptional activity. However,based on this comparison alone, it was difficult to predict on what thedifference in their properties depends.

On the other hand, the inventors have accumulated the results of thestudy in which the properties of DNA polymerases derived from a diverserange of organisms are reflected as those of chimeric Taq polymerases bythe following method: hot spring soil samples are collected from variousplaces, DNAs are directly extracted from the samples, fragments of DNApolymerase genes possessed by various kinds of organisms contained inthe samples are amplified by PCR based on the obtained DNAs(metagenomes), and the resulting fragments are recombined in vitro withthe homologous regions of Taq polymerase genes, whereby chimeric enzymesare constructed. It is, of course, ideal that the gene obtained from ametagenomic DNA contains a full-length sequence, but many metagenomicDNAs extracted from environmental samples are often fragmented ordamaged; thus, it is a very difficult task to obtain a full-length genedirectly. Therefore, we constructed the following study system; a genesegment encoding an active center that significantly affects theactivity of a DNA polymerase and peripheral regions thereof is obtainedfrom a metagenome, and the obtained segment is substituted with thehomologous region of a Taq polymerase gene to create a chimeric enzymegene, so that the obtained gene fragment could directly affect the basicproperties of a DNA polymerase.

We created a phylogenetic tree (not shown in the present application) bychecking the results of determining the activities of many chimeric Taqpolymerases constructed in such a way as described above, against eachother, and comparing the sequences of the gene fragments that arederived from metagenomic DNAs and which were introduced into wild-typeTaq polymerases. As a result of predicting the factors that might changethe activities of the chimeric Taq polymerases, we found the chimericTaq polymerases 8-16, 18-7, 1-8, and 3-7 which have significantly strongreverse transcriptional activity as compared with the wild-type Taqpolymerases.

First, we compared the sequences of 8-16, a Taq polymerase, and a Tthpolymerase with each other and, as a result, did not find any amino acidresidue that is common between 8-16 and the Tth polymerase but isdifferent between 8-16 and the Taq polymerase. However, among ten aminoacid residues that are different between the Taq and Tth polymerases,there were three amino acid residues that are completely different innature between these three polymerases. Thus, we focused on these threeamino acid residues and decided to introduce mutations to them.

Next, we compared the sequence of 18-7 with each of the sequences of aTaq polymerase and a Tth polymerase to thereby search for an amino acidresidue that is common between 18-7 and the Tth polymerase which bothhave reverse transcriptional activity but not common between 18-7 andthe Taq polymerase, and, as a result, four amino acid residues werefound. Among them, there was one amino acid residue that is greatlydifferent in nature between 18-7/Tth polymerase and the Taq polymerase;thus, we decided to introduce a mutation to this amino acid residue.

Further, as regards 1-8 and 3-7, these chimeric polymerases have strongprimer extension activity and reverse transcriptional activity, whereasthere was a chimeric enzyme (1-20) that has almost the same sequence assaid polymerases but is extremely weak in activity. We compared thesequences of the three enzymes: 1-8, 3-7 as well as 1-20 mentionedabove. As a result, we found two amino acids that are completelyinconsistent between these sequences, and selected the one that is onlyinconsistent in 1-20, which is greatly different in activity, anddecided to introduce a mutation to it.

We compared the mutant proteins of the site-specific mutated Taqpolymerase and Tth polymerase constructed according to the above-notedstrategy, with the wild-type enzymes. First, the mutant proteins wereevaluated for nucleotide incorporation activity using an activated DNAas a template, whereupon the respective specific activities of themutants which are relative to those of the wild-type Taq polymerasetaken as 100% demonstrated that there is in principle no significantchange in DNA polymerase activity. Next, these enzymes were determinedfor reverse transcriptional activity by a nucleotide incorporation assayusing RNA as a template, whereupon there were found a mutant Taqpolymerase (Taq R651E) and a mutant Tth polymerase (Tth P653E), whichrespectively have a reverse transcriptional activity 1.4 and 1.7 timesgreater than that of the wild-type Tth polymerase. Comparison of thesequences showed that the amino acid at position 653 in the Tthpolymerase corresponds to that at position 651 in the Taq polymerase(refer to FIG. 1). The inventors thus created mutant DNA polymeraseshaving presumably very useful properties, and completed the presentinvention.

The present invention provides the following:

[1] A DNA polymerase which is any one of (a) to (c) mentioned below:(a) a DNA polymerase comprising: an amino acid sequence modified fromthe amino acid sequence of SEQ ID NO: 12, which has a substitution ofthe arginine residue at position 651 by an amino acid residue having anegatively charged side chain; or an amino acid sequence that ismodified from an amino acid sequence of a Family A DNA polymerasederived from a thermophilic eubacterium, which has a substitution of anamino acid residue corresponding to position 651 in SEQ ID NO: 12 by anamino acid residue having a negatively charged side chain;(b) a DNA polymerase comprising an amino acid sequence modified from theamino acid sequence of the DNA polymerase as recited in (a), which has asubstitution, deletion, insertion and/or addition of one to nine aminoacid residues which exclude the amino acid residue substituted in (a);and(c) a DNA polymerase comprising an amino acid sequence that is at least95% identical to the amino acid sequence of the DNA polymerase asrecited in (a), with the proviso that the amino acid residue substitutedin (a) is the same as the amino acid residue in (a);[2] The DNA polymerase as recited in [1], wherein the Family A DNApolymerase derived from a thermophilic eubacterium is selected from thegroup consisting of DNA polymerases derived from Thermus aquaticus orDNA polymerases derived from Thermus thermophilus;[3] The DNA polymerase as recited in [11] or [2], wherein the DNApolymerase of (a) is:(a1) a DNA polymerase comprising an amino acid sequence modified fromthe amino acid sequence of SEQ ID NO: 12, which has a substitution ofthe arginine residue at position 651 by an amino acid residue having anegatively charged side chain;[4] The DNA polymerase as recited in [3], wherein the DNA polymerase of(a1) comprises the amino acid sequence of SEQ ID NO: 20;[5] The DNA polymerase as recited in [3] or [4], wherein the DNApolymerase of (b) or (c) has a reverse transcriptional activity of atleast 16.0×10³ U/mg;[6]A polynucleotide which is any one of (A1) to (D1) mentioned below:(A1) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:19;(B1) a polynucleotide comprising a nucleotide sequence encoding the DNApolymerase as recited in [3];(C1) a polynucleotide that hybridizes under stringent conditions with apolynucleotide comprising a complementary sequence to the nucleotidesequence of SEQ ID NO: 19, and which encodes a DNA polymerase; and(D1) a polynucleotide encoding a DNA polymerase comprising a sequence atleast 95% identical to the nucleotide sequence of SEQ FD NO: 19;[7] The polynucleotide as recited in [6], wherein the DNA polymerase in(C1) or (D1) has a reverse transcriptional activity of at least 16.0×10³U/mg;[8] The DNA polymerase as recited in [1] or [2], wherein the DNApolymerase of (a) is:(a2) a DNA polymerase comprising an amino acid sequence modified fromthe amino acid sequence of SEQ ID NO: 14, which has a substitution ofthe proline residue at position 653 by an amino acid residue having anegatively charged side chain;[9] The DNA polymerase as recited in [8], wherein the DNA polymerase of(a2) comprises the amino acid sequence of SEQ ID NO: 22;[10] The DNA polymerase as recited in [8] or [9], wherein the DNApolymerase of (b) or (c) has a reverse transcriptional activity of atleast 16.0×10³ U/mg;[11] A polynucleotide which is any one of (A2) to (D2) mentioned below:(A2) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21;(B2) a polynucleotide comprising a nucleotide sequence encoding the DNApolymerase as recited in [8];(C2) a polynucleotide that hybridizes under stringent conditions with apolynucleotide comprising a complementary sequence to the nucleotidesequence of SEQ ID NO: 21, and which encodes a DNA polymerase; and(D2) a polynucleotide encoding a DNA polymerase comprising a sequence atleast 95% identical to the nucleotide sequence of SEQ ID NO: 21;[12] The polynucleotide as recited in [11], wherein the DNA polymerasein (C2) or (D2) has a reverse transcriptional activity of at least16.0×10 U/mg;[13] A DNA polymerase which is any one of (a3) to (c3) mentioned below:(a3) a DNA polymerases comprising an amino acid sequence modified fromthe amino acid sequence of SEQ ID NO: 12, which has a substitution of atleast one selected from the glutamic acid residue at position 117, theasparatic acid residue at position 119, the asparatic acid residue atposition 142, and the asparatic acid residue at position 144 by an aminoacid residue having a non-polar aliphatic side chain;(b3) a DNA polymerase comprising an amino acid sequence modified fromthe amino acid sequence of the DNA polymerase as recited in (a3), whichhas a substitution, deletion, insertion and/or addition of one to nineamino acid residues, with the proviso that at least one amino acidresidue corresponding to at least one selected from positions 117, 119,142 and 144 in the amino acid sequence of the DNA polymerase as recitedin (a3) remains the same; and(c3) a DNA polymerase comprising an amino acid sequence that is at least95% identical to the amino acid sequence of the DNA polymerase asrecited in (a3), with the proviso that at least one amino acid residuecorresponding to at least one selected from positions 117, 119, 142 and144 in the amino acid sequence of the DNA polymerase as recited in (a3)remains the same;[14] The DNA polymerase as recited in [13], wherein the DNA polymeraseof (a3) comprises the amino acid sequence of SEQ ID NO: 16;[15] The DNA polymerase as recited in 113) or [14], wherein the DNApolymerase of (b3) or (c3) has a primer extension activity using DNA asa template, of at least 4.00 kb/U·min.[16]A polynucleotide which is any one of (A3) to (D3) mentioned below:(A3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15;(B3) a polynucleotide comprising a nucleotide sequence encoding the DNApolymerase as recited in 1:13);(C3) a polynucleotide that hybridizes under stringent conditions with apolynucleotide comprising a complementary sequence to the nucleotidesequence of SEQ ID NO: 15, and which encodes a DNA polymerase; and(D3) a polynucleotide encoding a DNA polymerase comprising a sequence atleast 95% identical to the nucleotide sequence of SEQ ID NO: 15;[17] The polynucleotide as recited in [16], wherein the DNA polymeraseof (C3) or (D3) has a primer extension activity using DNA as a template,of at least 4.00 kb/U·min.[18]A DNA polymerase which is any one of (a4) to (c4) mentioned below:(a4) a DNA polymerases comprising an amino acid sequence modified fromthe amino acid sequence of SEQ ID NO: 12, which has a substitution of atleast one selected from the glutamic acid residue at position 117, theasparatic acid residue at position 119, the asparatic acid residue atposition 142, and the asparatic acid residue at position 144, as well asthe glutamic acid residue at position 742 by an amino acid residuehaving a non-polar aliphatic side chain;(b4) a DNA polymerase comprising an amino acid sequence modified fromthe amino acid sequence of the DNA polymerase as recited in (a4), whichhas a substitution, deletion, insertion and/or addition of one to nineamino acid residues, with the proviso that amino acid residuescorresponding to at least one selected from positions 117, 119, 142 and144, as well as position 742, in the amino acid sequence of the DNApolymerase as recited in (a4) remain the same; and(c4) a DNA polymerase comprising an amino acid sequence that is at least95% identical to the amino acid sequence of the DNA polymerase asrecited in (a4), with the proviso that amino acid residues correspondingto at least one selected from positions 117, 119, 142 and 144, as wellas position 742, in the amino acid sequence of the DNA polymerase asrecited in (a4) remain the same;[19] The DNA polymerase as recited in [18], wherein the DNA polymeraseof (a4) comprises the amino acid sequence of SEQ ID NO: 18;[20] The DNA polymerase as recited in 1181 or [19], wherein the DNApolymerase of (b4) or (c4) has a primer extension activity using DNA asa template, of at least 4.00 kb/U·min.[21] A polynucleotide which is any one of (A4) to (D4) mentioned below:(A3) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17;(B4) a polynucleotide comprising a nucleotide sequence encoding the DNApolymerase as recited in [18];(C4) a polynucleotide that hybridizes under stringent conditions with apolynucleotide comprising a complementary sequence to the nucleotidesequence of SEQ ID NO: 17, and which encodes a DNA polymerase; and(D4) a polynucleotide encoding a DNA polymerase comprising a sequence atleast 95% identical to the nucleotide sequence of SEQ ID) NO: 17;[22] The polynucleotide as recited in [21], wherein the DNA polymeraseof (C4) or (D4) has a primer extension activity using DNA as a template,of at least 4.00 kb/U·min.[23]A recombinant vector comprising the polynucleotide as recited in anyone of [7], [12], [17] and [22];[24]A transformant comprising the recombinant vector as recited in [23];and[25]A process for preparing the DNA polymerase as recited in any one of[1] to [5], [8] to [10], [13] to [15], and [18] to [20], the processcomprising a step of culturing the transformant as recited in [24].

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1-1 shows a comparison and alignment of the amino acid sequences ofTaq and Tth DNA polymerases. The mark “∇” indicates the correspondencebetween the amino acid at position 651 in the Taq DNA polymerase and theamino acid at position 653 in the Tth DNA polymerase.

FIG. 1-2 shows a comparison and alignment of the amino acid sequences ofTaq DNA polymerase and other family A DNA polymerases from thermophilicbacteria. The mark “▾” indicates the amino acid at position 651 in theTaq DNA polymerase.

FIG. 1-3 shows a comparison and alignment of the amino acid sequences ofTaq DNA polymerase and other family A DNA polymerases from thermophilicbacteria. The mark “▾” indicates the amino acid at position 651 in theTaq DNA polymerase.

FIG. 2 shows a photograph of the SDS-PAGE gels concerning purificationof Taq DNA polymerase mutants. The purity of each of the prepared TaqDNA polymerase mutants was confirmed by SDS-PAGE analysis (refer toExample 1).

FIG. 3 shows a graph of an actual example of enzymatic activitydetermination by nucleotide incorporation assay (refer to Example 2).Under the conditions of this test, Taq showed an activity of 3.9×10⁵U/mg.

FIG. 4 shows an example of a graph for determining primer extensionrate. With the amount of enzyme kept constant, a time course of primerextension reaction is taken and the extension rate can be determined bymeasuring the length of strand extended per unit time through alkalineagarose electrophoresis in a region where the plots lie on a straightline (refer to Example 2). Under the conditions of this test, Taq showeda rate of 4.67 kb/min/U, Taq′ (Taq mutant) showed a rate of 6.67kg/min/U, and Chimera A (the chimera created by recombining a segment ofthe Taq gene with a homologous region obtained from the metagenome)showed a rate of 11.20 kb/min/U.

FIG. 5 shows a photograph of the alkaline agarose electrophoresis gelsfor comparing primer extension rates.

FIG. 6 shows the Taq WT amino acid sequence of SEQ ID) NO: 12 and theTaq WT nucleotide sequence of SEQ ID NO: 11.

FIG. 7 shows the Tth WT amino acid sequence of SEQ ID NO: 14 and the TthWT nucleotide sequence of SEQ ID NO: 13.

FIG. 8 shows the Taq Exo⁻ amino acid sequence of SEQ ID NO: 16 and theTaq Exo⁻ nucleotide sequence of SEQ ID NO: 15.

FIG. 9 shows the Taq Exo⁻+E742A amino acid sequence of SEQ ID NO: 18 andthe Taq Exo⁻+E742A nucleotide sequence of SEQ ID NO: 17.

FIG. 10 shows the Taq R651E amino acid sequence of SEQ ID NO: 20 and theTaq R651E nucleotide sequence of SEQ ID NO: 19.

FIG. 11 shows the Tth P653B amino acid sequence of SEQ ID NO: 22 and theTth P653E nucleotide sequence of SEQ ID NO: 21.

DESCRIPTION OF EMBODIMENTS Definitions, etc.

The “DNA polymerase” as referred to in the present invention, unlessotherwise specified, means a protein having the activity of extending acomplementary DNA strand to a template nucleic acid (DNA or RNA) usingdeoxyribonucleoside triphosphate as a substrate. The “Taq polymerase” or“Taq DNA polymerase” as referred to in this invention, unless otherwisespecified, means a DNA polymerase derived from Thermus aquaticus. Theamino acid sequence and the nucleotide sequence are respectively shownin SEQ ID NOs: 12 and 11 in the Sequence Listing, which constitutes apart of the present specification. The “Tth polymerase” or “Tth DNApolymerase” as referred to in this invention means a DNA polymerasederived from Thermus thermophilus. The amino acid sequence and thenucleotide sequence are respectively shown in SEQ ID NOs: 14 and 13 inthe Sequence Listing, which constitutes a part of the presentspecification.

DNA Polymerase Activity:

The “activity” as referred to in the present invention in connectionwith a DNA polymerase includes transcriptional activity and primerextension activity.

The transcriptional activity includes transcriptional activity using DNAas a template, and transcriptional activity using RNA as a template. Aswell known to those skilled in the art, the transcriptional activity canbe determined as the activity of incorporating deoxyribonucleosidetriphosphate (dNTP) as a substrate. More specifically, a calf thymusDNA, a salmon sperm DNA, or the like is partially digested with DNase 1to provide a nicked or gapped double-strand DNA as a template, and aradioisotope-labeled dNTP is mixed with a substrate dNTP; then, the DNApolymerase of interest is caused to act, so that the amount ofnucleotides incorporated into nicks by nick translation or into gaps byprimer extension activity can be determined using radioactivity as anindicator. This determination method, which is called nucleotideincorporation assay, is a standard method for determining DNA polymeraseactivity.

The “transcriptional activity” or “basic DNA polymerase activity” asreferred to in the present invention, unless otherwise specified, meansthe activity of incorporating dNTP when DNA strand is used as atemplate. When the “transcriptional activity” or “basic DNA polymeraseactivity” is represented by numerical value in this invention, unlessotherwise specified, the amount of enzyme, i.e., DNA polymerase,required to incorporate 10 nmol of nucleotides at 72° C. for 30 minutesis defined as 1 unit (U), and such activity is expressed as a value forspecific activity (activity per protein amount) in U/mg or the like.Unless otherwise specified, the conditions for this determination areset as disclosed in the Examples section of the present application.

The “reverse transcriptional activity” as referred to in the presentinvention, unless otherwise specified, means the activity ofincorporating dNTP when RNA strand is used as a template. When the“reverse transcriptional activity” is represented by numerical value inthis invention, unless otherwise specified, the amount of enzyme, i.e.,DNA polymerase, required to incorporate 10 nmols of nucleotide at 72° C.for 30 minutes is defined as 1 unit (U), and such activity is expressedas a value for specific activity (activity per protein amount) in U/mgor the like. Unless otherwise specified, the conditions for thisdetermination are set as disclosed in the Examples section of thepresent application.

The “primer extension activity” as referred to in the present invention,unless otherwise specified, means the length of strand extended per unittime when a DNA polymerase of interest is caused to act on a substratedNTP using DNA or RNA as a template, and the length can be expressed inkb/U/min, bp/pmol/min, or the like. Unless otherwise specified, theconditions for this determination are set as disclosed in the Examplessection of the present application.

DNA Polymerases of the Present Invention:

The present invention provides novel DNA polymerases, more specificallymutants of Family A DNA polymerases derived from thermophiliceubacteria, notably particular mutants of Thermus aquaticus polymerases(Taq polymerases) or mutants of homologues thereof.

The “thermophilic eubacteria” as referred to herein means eubacteriahaving an optimum growth temperature of at least 45° C. or at least 60°C. Examples include bacteria of the genus Thermus, such as Thermusaquaticus and Thermus thermophilus, those of the genus Thermotoga, suchas Thermotoga maritima, those of the genus Aquifex, such as Aquifexaeolicus, and those of the genus Thermodesulfobacerium, such asThermodesulfobacterium commune, with preference given to Thermusaquaticus and Thermus thermophilus.

Thus, examples of the DNA polymerase of the present invention that arepreferably provided include not only mutants of Thermusaquaticus-derived DNA polymerases (Taq polymerases) or mutants ofThermus thermophilus-derived DNA polymerases (Tth polymerases), but alsomutants of Family A DNA polymerases derived from other thermophiliceubacteria, in which an amino acid that is shown by sequence comparisonto correspond to a mutation site of a Taq polymerase is mutated.

For example, it was found that when the amino acid sequences of aThermus aquaticus-derived DNA polymerase (Taq polymerase) and a Thermusthermophilus-derived DNA polymerase (Tth polymerase) are compared andaligned, the amino acid at position 651 in the Taq polymerase and theamino acid at position 653 in the Tth polymerase correspond to eachother (FIG. 1).

As a result of investigation about chimeric Taq polymerases, the presentinventors found two chimeric enzymes having high primer extensionactivity and reverse transcriptional activity, and other chimericenzymes which have almost the same sequences as said two enzymes butshow extremely low activities. The inventors also selected from thosesequences one residue that is only inconsistent in the low-activityenzymes, and introduced a mutation to this residue, whereby theinventors found a mutant Taq polymerase (Taq R651E) and a mutant Tthpolymerase (Tth P653E), which respectively have a reversetranscriptional activity 1.4 and 1.7 times greater than that of thewild-type Tth polymerase.

Thus, in the first aspect, the present invention provides a DNApolymerase comprising an amino acid sequence modified from an amino acidsequence of a Family A DNA polymerase derived from a thermophiliceubacterium, which has a substitution of one corresponding amino acidresidue by an amino acid residue having a negatively charged side chain(in respect of the first aspect, refer to [1] and [2] mentioned above).Specifically, this invention provides a DNA polymerase comprising: anamino acid sequence modified from the amino acid sequence of SEQ ID NO:12, which has a substitution of the arginine residue at position 651 byan amino acid residue having a negatively charged side chain; or anamino acid sequence modified from an amino acid sequence of a Family ADNA polymerase derived from a thermophilic eubacterium, which has asubstitution of an amino acid residue corresponding to position 651 inSEQ ID NO: 12 by an amino acid residue having a negatively charged sidechain (in respect of the first aspect, refer to [1] and [2] mentionedabove). More specifically, this invention provides a DNA polymerasecomprising the mutant Taq R651E or a homologue thereof, or the mutantTth P653E or a homologue thereof (in respect of the first aspect, referto [3] to [7] mentioned above for the mutant Taq R651E, and [8] to [12]for mutant Tth P653E).

The amino acid sequence of Taq R651E and the nucleotide sequenceencoding the same are respectively shown in SEQ ID NOs: 20 and 19 in theSequence Listing which constitutes a part of the present specification.The amino acid sequence of Tth P653E and the nucleotide sequenceencoding the same are respectively shown in SEQ ID) NOs: 22 and 21 inthe Sequence Listing which constitutes a part of the presentspecification.

The DNA polymerase according the first aspect of the present inventionis characterized by having improved reverse transcriptional activityover the wild-type DNA polymerase. To be specific, the mutant Taq R651Eand the homologues thereof have a reverse transcriptional activity of atleast 5.00×10³ U/mg, preferably at least 10.0×10³ U/mg, more preferablyat least 1.5.0×10³ U/mg, and still more preferably at least 20.0×10³(U/mg. The mutant Tth P653E and the homologues thereof have a reversetranscriptional activity of at least 16.0×10³ U/mg, preferably at least18.0×10³ U/mg, and more preferably at least 20.0×10³ U/mg.

The present inventors further made extensive studies for the purpose ofcreating a superior PCR enzyme by modifying a Taq polymerase, and as aresult found that the mutant Taq Exo⁻ (E117A, D119A, D142A, D144A), inwhich four amino acid residues presumably important for the 5′→3′exonuclease activity inherent in a Taq polymerase are converted at thesame time, are superior to the wild-type Taq polymerase in terms ofprimer extension activity using DNA as a template. Thus, in the secondaspect, this invention provides the mutant Taq Exo⁻ and homologuesthereof, polypeptides encoding the same and homologues thereof (inrespect of the second aspect, refer to [13] to [17] mentioned above).

The amino acid sequence of Taq Exo⁻ and the nucleotide sequence encodingthe same are respectively shown in SEQ ID NOs: 16 and 15 in the SequenceListing which constitutes a part of the present specification.

In Taq Exo⁻, all of the glutamic acid residue at position 117, theasparatic acid residue at position 119, the asparatic acid residue atposition 142, and the asparatic acid residue at position 144 in theamino acid sequence of SEQ ID NO: 12 are substituted, but the homologuesof said mutant, which have an amino acid sequence in which at least one,preferably two, more preferably three, selected from these residues areeach independently substituted by an amino acid having a non-polaraliphatic side chain, preferably by an amino acid selected from thegroup consisting of glycine, alanine, valine, leucine, isoleucine andproline, and more preferably by alanine, are also encompassed by thepresent invention.

The mutant Taq Exo⁻ and the homologues thereof according to the secondaspect of the present invention have a primer extension activity usingDNA as a template, at least 4.00 kb/U·min, preferably at least 8.00kb/U·min, more preferably 9.00 kb/U·min, and still more preferably 14.0kb/U·min.

Furthermore, the present inventors found that the mutant Taq Exo⁻ towhich the mutation of Patent Document 3 mentioned above is furtherintroduced achieves further improvement in extension activity. Thus, inthe third aspect, the present invention provides the mutant TaqExo⁻+E742A and homologues thereof; polypeptides encoding the same andhomologues thereof (in respect of the third aspect, refer to [18] to[22] mentioned above).

The amino acid sequence of Taq Exo⁻+E742A and the nucleotide sequenceencoding the same are respectively shown in SEQ ID NOs: 18 and 17 in theSequence Listing, which constitutes a part of the present specification.

In Taq Exo⁻+E742A, all of the glutamic acid residue at position 117, theasparatic acid residue at position 119, the asparatic acid residue atposition 142, and the asparatic acid residue at position 144, as well asthe glutamic acid residue at position 742, in the amino acid sequence ofSEQ ID NO: 12 are substituted, but the homologues of said mutant, whichhave an amino acid sequence in which at least one, preferably two, morepreferably three, still more preferably four, selected from theseresidues are each independently substituted by an amino acid having anon-polar aliphatic side chain, preferably by an amino acid selectedfrom the group consisting of glycine, alanine, valine, leucine,isoleucine and proline, and more preferably by alanine, are alsoencompassed by the present invention.

The mutant Taq Exo⁻+E742A and the homologues thereof according to thethird aspect of the present invention have a primer extension activityusing DNA as a template, at least 4.00 kb/U·min, preferably at least8.00 kb/U·min, more preferably 9.00 kb/U·min, and still more preferably14.0 kb/U·min.

Any of the above-mentioned mutants of the present invention encompassestheir variants in which a segment having exonuclease activity andconsisting of multiple consecutive amino acid residues starting from theN terminal side is deleted. The N-terminal segment that may be deletedcan be designed as appropriate by those skilled in the art. In the caseof mutant Taq polymerases, the segment typically consists of theresidues at positions 1-233, positions 1-293, or positions 1-302, in theamino acid sequence of SEQ ID NO: 12. In the case of mutant Tthpolymerases, the segment typically consists of the residues at positions1-237 in the amino acid sequence of SEQ ID NO: 14, which correspond topositions 1-233 in the Taq sequence. In every case, the mutant DNApolymerase of this invention even encompasses those variants in which asegment of a shorter amino acid residue length than in theabove-mentioned deletion variants is deleted as long as the length iswithin the segment ranges mentioned above.

When the phrase “which has a substitution, deletion, insertion and/oraddition of one to nine amino acid residues” is used in the presentinvention, the number of amino acids to be substituted or otherwisemodified is not particularly limited as long as the protein (DNApolymerase) having the modified amino acid sequence has a desiredfunction, and 1-9 or about 1-4 amino acids may be substituted orotherwise modified, or even more amino acids may be substituted orotherwise modified if said substitution or the like is intended toencode the same or similar amino acid sequence. Means for obtaining aprotein having such an amino acid sequence are well known to thoseskilled in the art.

The substitution or the like of an amino acid(s) may be such asubstitution or the like that does not cause an electrostatic change,for example, a substitution by an amino acid(s) that is(are) similar inelectric charge and/or polarity. Examples of such a substitutioninclude: substitution between amino acids having such an aliphatic sidechain that the side chain (also expressed as “R group”) is non-polararound physiological pH (7.0) (e.g., glycine, alanine, valine, leucine,isoleucine, and proline); substitution between amino acids having apolar uncharged side chain (e.g., serine, threonine, cysteine,methionine, asparagine, and glutamine); substitution between amino acidshaving a side chain that is positively charged around physiological pH(e.g., lysine, arginine, and histidine); substitution between aminoacids having a negatively charged side chain (e.g., asparatic acid,glutamic acid); substitution between polar amino acids; and substitutionbetween non-polar amino acids.

Examples of DNA polymerases preferred in the present invention includenot only the mutant DNA polymerases encompassed by the above-mentionedfirst to third aspects of the invention, but also variants of wild-typeTaq polymerases (SEQ ID NO: 12) or wild-type Tth polymerases (SEQ ID NO:14) which have such an amino acid substitution as characterized above atposition 651, 664, 674 or the like. Specific examples include a mutantof a wild-type Taq polymerase (SEQ ID NO; 12), which has a substitutionof an arginine residue at position 651 by a glutamic acid residue, amutant of the same polymerase having a substitution of a threonineresidue at position 664 by an arginine residue, and a mutant of the samepolymerase having a substitution of a serine residue at position 674 byan arginine residue (refer to FIG. 5).

The “stringent conditions” as referred to in the present invention canbe determined as appropriate by those skilled in the art on the basis ofthe reference data such as polynucleotide length. As to the stringentconditions, those skilled in the art can make reference to theconditions described in Sambrook, et al., Molecular Cloning: ALaboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press,2001). The stringent conditions refer to, for example, the hybridizationconditions of about 50% formamide, 2×SSC to 6×SSC at about 40-50° C. (orthose conditions for other similar hybridization solutions such asStark's solution in about 50% formamide at about 42° C.). Thepre-washing conditions of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0), and/orthe washing conditions of 0.5×SSC, 0.1% SDS at about 60° C. may also beapplied. The stringent conditions more preferably involve not only thehybridization conditions of about 50% formamide, 2×SSC to 6×SSC at about40-50° C. (or those conditions for other similar hybridization solutionssuch as Stark's solution in about 50% formamide at about 42° C.), butalso the washing conditions of 0.2×SSC, 0.1% SDS at about 68° C.

The high identity as referred to in the present invention in connectionwith an amino acid sequence, unless otherwise specified, means asequence identity of at least 80%, preferably at least 90%, morepreferably at least 95%, still more preferably at least 96%, and mostpreferably at least 97%. The high identity as referred to in the presentinvention in connection with a nucleotide sequence, unless otherwisespecified, also means a sequence identity of at least 80%, preferably atleast 90%, more preferably at least 95%, still more preferably at least96%, and most preferably 97%. Search and analysis of polynucleotide oramino acid sequence identity can be made using an algorithm or programwell known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX,ClustalW). When the program is used, parameters can be appropriately setby those skilled in the art, or the default parameters of each programmay also be used. Specific procedures for such analyses are also wellknown to those skilled in the art.

Of both amino acid and nucleotide sequences, an important site forperformance of an intended function is described in the presentspecification. Accordingly, those skilled in the art can design, prepareand use different mutants in which the sequences of other segments thansuch an important site are modified as appropriate. Such mutants canalso fall within the scope of the present invention.

The DNA polymerases of the present invention can be prepared by a methodwell known to those skilled in the art. In order to construct arecombinant vector, a DNA fragment of an appropriate length, whichcontains the coding region of a protein of interest, is prepared as afirst step. In the nucleotide sequence of the coding region of theprotein of interest, nucleotides may be so substituted as to giveoptimal codons for expression in host cells. Next, the prepared DNAfragment is inserted downstream of a promoter in an appropriateexpression vector to construct a recombinant vector. It is necessarythat said DNA fragment be incorporated into the vector so as to performits function. The vector may contain not only promoters but also ciselements (e.g., enhancers), splicing signals, polyadenylation signals,selective markers (e.g., dihydrofolate reductase gene, ampicillinresistance gene, neomycin resistance gene), ribosome binding sequences(SD sequences), and/or the like. A transformant capable of producing aprotein of interest can be obtained by introducing a recombinant vectorinto an appropriate host cell.

The expression vector is not particularly limited as long as it iscapable of autonomous replication in a host cell, and examples of thevector that can be used include plasmid vectors, phage vectors, andviral vectors. Examples of the plasmid vectors that can be used includeE. coli-derived plasmids (e.g., pRSET, pBR322, pBR325, pUC118, pUC119,pUC18, and pUC19), Bacillus subtilis-derived plasmids (e.g., pUB110 andpTPS), and yeast-derived plasmids (e.g., YEp13, YEp24, and YCp50).Examples of the phage vectors that can be used include λ phages (e.g.,Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, and λZAP). Examples ofthe viral vectors that can be used include animal viruses such asretroviruses and vaccinia viruses, and insect viruses such asbaculoviruses.

As the host cell, there can be used any of prokatyocytes, yeasts, animalcells, insect cells, plant cells, and other cells, as long as the cellis capable of expressing a DNA encoding a protein of interest. Animalindividuals, plant individuals, silkworms, and the like may also beused.

When a bacterium is used as a host cell, examples of the bacterium thatcan be used as a host cell include bacteria of the genus Escherichia,such as Escherichia coli, those of the genus Bacillus, such as Bacillussubtilis, those of the genus Pseudomonas, such as Pseudomonas putida,and those of the genus Rhizobium, such as Rhizobium meliloti. Specificexamples of the bacterium that can be used as a host cell includeEscherichia coli such as Escherichia coli BL21, Escherichia coliXL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichiacoli K 12, Escherichia coli JM109, and Escherichia coli HB101, andBacillus subtilis such as Bacillus subtilis MI 114, and Bacillussubtilis 207-21. The promoter used in this case is not particularlylimited as long as it can be expressed in a bacterium such as E. coli,and examples of the promoter that can be use include those derived fromE. coli, phages and the like, such as trp promoter, lac promoter, P_(L)promoter, and P_(R) promoter. Artificially designed and modifiedpromoters such as tac promoter, lacT7 promoter, and let 1 promoter canalso be used. When a yeast is used as a host cell, examples of the yeastthat can be used as a host cell include Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Pichia pastoris. The promoter used inthis case is not particularly limited as long as it can be expressed ina yeast, and examples of the promoter that can be used include gallpromoter, gal10 promoter, heat shock protein promoter, MFα1 promoter,PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, and AOX1promoter. When an insect cell is used as a host, examples of the insectcell that can be used as a host cell include Spodoptera frugiperdaovarian cells, Trichoplusia ni ovarian cells, and cultured cells derivedfrom silkworm ovaries. Examples of the Spodoptera frugiperda ovariancells that can be used include Sf9) and Sf21; examples of theTrichoplusia ni ovarian cells that can be used include High 5 andBTI-TN-5B1-4 (Invitrogen); and examples of the cultured cells derivedfrom silkworm ovaries that can be used include Bombyx mori N4.

The method for introducing a recombinant vector into a host is notparticularly limited, as long as the method allows introduction of a DNAinto the host, and examples of the method that can be used include acalcium ion method, electroporation, a spheroplast method, and a lithiumacetate method. The method for introducing a recombinant vector into aninsect cell is not particularly limited, as long as the method allowsintroduction of a INA into the insect cell, and examples of the methodthat can be used include a calcium phosphate method, lipofection, andelectroporation.

A transformant having introduced therein a recombinant vectorincorporating a DNA encoding a protein of interest is cultured.Culturing of the transformant can be carried out according to aconventional method used for culture of host cells.

The protein of interest can be obtained by collecting it from theculture of the transformant. The “culture” as referred to hereinencompasses all of culture supernatants, cultured cells, culturedmicroorganisms, and disrupted products of cells or microorganisms. Inthe case where the protein of interest is accumulated in transformantcells, the culture is centrifuged to collect the cells from the culture,and the collected cells are washed and disrupted to extract the proteinof interest. In the case where the protein of interest is excretedoutside the transformant cells, the culture supernatant is used as itis, or cells or microorganisms are removed from the culture supernatantby centrifugation or the like. The extracted protein can be purified bysolvent extraction, salting-out/desalting with ammonium sulfate or thelike, precipitation with an organic solvent, diethylaminoethyl(DEAE)-sepharose, ion exchange chromatography, hydrophobicchromatography, gel filtration, affinity chromatography, or the like.

The mutants of the present invention are useful for DNA amplification(particularly, PCR) and can be used as a component of a DNAamplification (particularly, PCR) kit. In addition to the inventivemutant Taq polymerases or fragments thereof, the DNA amplification(particularly, PCR) kit can contain reagents (e.g., four types of dNTPs,Mg²⁺, buffer, additives), a vessel, an apparatus, and the like which arenecessary for DNA amplification (particularly, PCR). The inventive DNAamplification method and kit are suitable for a variety of applications,including DNA sequencing, gene diagnosis, individual identification inpaternity test and criminal investigation, variety identification, SNP(single nucleotide polymorphism) analysis for constitutional study, andarchaeological excavation.

EXAMPLES Example 1 Construction of Mutants 1. Introduction of Mutations:

In order to construct each of amino acid substitution mutants of a Taqpolymerase, one or more site-specific mutations were introduced in aprimer dependent manner by PCR using one or more sets of primers havingsequences designed to ensure that a mutation is applied to a position ofinterest, with an expression plasmid having a Taq polymerase geneinserted therein being used as a template. The introduction of one ormore site-specific mutations into a Taq polymerase was performed usingone or more sets of primers shown below. The amino acid substitutionsites employed to construct the respective mutants are underlined.

[Formula 1] Exo Taq117119A-F (SEQ ID NO: 1)CTCGAGGTCCCGGGCTACGCGGCGGCCGACGTCCTGGCCAGCCTG Taq117119A-R(SEQ ID NO: 2) CACGCTGGCCAGGACGTCGGCCGCCGCGTAGCCGGGACCTCGAG Taq142144A-F(SEQ ID NO: 3) GTCCGCATCCTCACCGCCGCCAAAGCCCTTTACCAGCTCCTTTCCTaq142144A-R (SEQ ID NO: 4)GGAAAGGAGCTGGTAAAGGGCTTTGGCGGCGGTGAGGATGCGGAC [Formula 2] Exo + E742ATaq117119A-F (SEQ ID NO: 1)CTCGAGGTCCCGGGCTACGCGGCGGCCGACGTCCTGGCCAGCCTG Taq117119A-R(SEQ ID NO: 2) CAGGCTGGCCAGGACGTCGGCCGCCGCGTAGCCCGGGACCTCGAGTaq142144A-F (SEQ ID NO: 3)GTCCGCATCCTCACCGCCGCCAAAGCCCTTTACCAGCTCCTTTCC Taq142144A-R(SEQ ID NO: 4) GGAAAGGAGCTGGTAAAGGGCTTTGGCGGCGGTGAGGATGCGGAC E742A-F(SEQ ID NO: 5) GTGAAGAGCGTGCGGGCGGCGGCCGAGCGCATG E742A-R (SEQ ID NO: 6)CATGCGCTCGGCCGCCGCCCGCACGCTCTTCAC [Formula 3] TaqR651E R651E-F(SEQ ID NO: 7) ATGTTCGGCGTCCCCGAGGAGGCCGTGGAC R651E-R (SEQ ID NO: 8)GTCCACGGCCTCCTCGGGGACGCCGAACAT [Formula 4] TthP653E P653E-F(SEQ ID NO: 9) ATGTTCGGCGTGCCCGAGGAGGCCGTGGAC P653E-R (SEQ ID NO: 10)GTCCACGGCCTCCTCGGGGACGCCGAACAT

Fifty microliters of a PCR reaction mixture (20 ng of pTV-Taq plasmidDNA, 0.5 μM each primer set, 0.2 mM dNTP, and 1 U of Pyrobest DNApolymerase (Takara Bio)) was subjected to initial denaturation in aPyrobest buffer at 98° C. for 10 seconds, which was followed by PCRunder the conditions of 16 cycles (98° C. for 10 seconds, 55° C. for 30seconds, and 72° C. for 8 minutes). 5 U of the restriction enzyme Dpn1was added to the resulting PCR product, and the mixture was incubated at37° C. for 2 hours. Then, the reaction mixture was introduced into theE. coli JM109 strain, and the resulting strain was cultured. By usingthe procedure described in the next section, a plasmid was extractedfrom the resulting transformant clone, and then a check was made to seethat the mutation(s) was(were) introduced in the position(s) ofinterest.

2. Preparation of Plasmids and Confirmation of Nucleotide Sequences:

With a drug resistance gene in the plasmid being used as a marker,selection was made of an E. coli transformant that was seeded onto an LBplate medium containing 50 μg/mL of ampicillin and cultured at 37° C.for 15 hours. The colony that has grown was inoculated into 4 mL of anLB liquid medium containing 50 μg/mL of ampicillin and cultured at 37°C. for 15 hours. A plasmid was extracted from the harvestedmicroorganisms using a QIAprep Spin Miniprep Kit (QIAGEN) according tothe kit's protocol. With the DNA of the resulting plasmid being used asa template, dideoxy reaction was performed using a DTCS Quick StartMaster Mix (Beckman Coulter), so that the nucleotide sequence wasconfirmed using a multi-capillary DNA analysis system CEQ2000XL (BECKMANCOULTER).

The novel DNA polymerases constructed in the present study are listed inthe following table.

TABLE 1 DNA number of Theoretical Polymerase amino acids pl Mw.characteristics Taq WT (SEQ ID NO: 12) 832 6.04 93910.1 Taq Pol wildtype Taq8 E742A 832 6.06 93852.1 E742A Taq8 A743H 832 6.10 93976.2 A743H294L WT 540 6.88 60914.1 Deletion (293 as of N-ter) 294L E742A 540 6.9760856.1 Deletion (293 as of N-ter), E742A 303E WT 531 6.07 59896.9Deletion (302 as of N-ter) 303E E742A 531 6.18 59838.9 Deletion (302 asof N-ter), E742A Exo

WT (SEQ ID NO: 16) 832 6.31 93720.0 E117A, D119A, D142A, D144A Exo

E742A (SEQ ID NO: 18) 832 6.31 93662.0 E117A, D119A, D142A, D144A, E742ATaq R651E (SEQ ID NO: 20) 832 6.92 94785.2 R651E Taq T664R 832 6.1093965.2 T664R Taq G668R 832 6.10 94009.2 G668R Taq S674R 832 6.1093979.2 S674R Tth WT (SEQ ID NO: 14) 834 6.30 94049.4 Tth Pol Wild typeTth P653E (SEQ ID NO: 22) 834 6.23 94081.4 Tth Pol P653E

indicates data missing or illegible when filed

3. Expression and Purification of DNA Polymerases:

Production of each of wild-type and mutant Taq polymerases was performedunder the following conditions: the JM109 strain was transformed by astandard method using a plasmid that incorporated the gene of eachpolymerase into the pTV-118N vector, and the resulting transformant wascultured at 37° C. for 24 hours in 500 mL of an LB liquid mediumcontaining 50 μg/mL of ampicillin. Production of each of wild-type andmutant Tth polymerases was performed under the following conditions; theBL21 (DE3) CodonPlus-RIPL strain (Stratagene) was transformed by astandard method using pET-3C as an expression vector, and the resultingtransformant was cultured at 37° C. for 24 hours in 500 mL of an LBliquid medium containing 50 μg/mL of ampicillin and 331 μg/mL ofchloramphenicol. Thereafter, the culture was centrifuged at 6,000 rpmfor 15 minutes to harvest microorganisms, and the harvestedmicroorganisms were suspended in 25 mL of Buffer A (50 mM Tris-HCl (pH8.0), 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol) supplemented with 1 mMPMSF, and were subjected to ultrasonication and centrifuged at 14,500rpm for 15 minutes to obtain a crude cell extract. The crude extract wasleft to stand at 80° C. for 20-30 minutes to denature a non-thermostableprotein, and was centrifuged at 14,500 rpm for 15 minutes to obtain athermostable fraction in the supernatant. Polyethyleneimine was added tothe fraction on ice so as to give a concentration of 0.15%, and theprecipitate (nucleic acid) was removed by centrifugation at 14,500 rpmfor 15 minutes. Next, ammonium sulfate was added to the supernatant onice so as to give 80% saturation, and the suspension was stirred for atleast 1 hour to effect salting-out. The suspension was centrifuged at14,500 rpm for 15 minutes to effect protein precipitation, and theprecipitate was suspended in Buffer A supplemented with 0.8 M ammoniumsulfate; thereafter, the suspension was subjected to chromatography bypassing it through a Hi Trap Phenyl column (5 mL) using an ÅKTA Explorer(GE Healthcare). After passage of the sample, a gradient from 1 M to 0 Mammonium sulfate was created and ultrapure water was passed through thecolumn to thereby elute an enzyme of interest. The fraction wasrecovered and passed through a Hi Trap Heparin column (1 mL). Elutionwas performed with a gradient from 0 M to 800 mM sodium chloride asdissolved in Buffer A. The enzymes of interest thus obtained were eachanalyzed by SDS-PAGE to confirm their purity.

Example 2 Evaluation of Mutants 1. Transcriptional Activity:

In order to determine the basic DNA polymerase activity of each of thepurified enzymes, a world-wide standard method was used. Morespecifically, the intensity of the activity of incorporatingdeoxyribonucleotides on a DNA template strand was determined, and theactivity per unit protein amount was calculated in units. To perform anucleotide incorporation reaction, a reaction mixture was prepared byadding 0.2 mg/mL of activated DNA (obtained by treating a calf thymusDNA with DNase I to partially nick or gap a double-strand DNA), 0.2 mMdNTP, 440 nM [³H]-dTTP, 50 mM Tris-HCl (pH 8.0), 1.5 mM MgCl₂, 50 mMKCl, 0.1% TritonX-100, 100 μg/mL of BSA, and 1 nM DNA polymerase, andthe mixture was reacted at 72° C.; then, 10 μL of the mixture wasspotted onto DE81 paper. After air-dried for 10 minutes, the paper waswashed with an aqueous 5% disodium hydrogenphosphate solution to removeunreacted nucleotides. The washing was repeated three times each for 10minutes. After the DE81 paper was dried, radiation was measured by aliquid scintillation counter, whereby the amount of (³H-dTMPincorporated in the activated DNA due to the DNA polymerase activity wascalculated to determine enzyme activity (in unit). One unit is definedas the amount of enzyme, i.e., DNA polymerase, required to incorporate10 nmol of nucleotides at 72° C. for 30 minutes. The specific activitywas calculated for each enzyme.

2. Extension Activity (Extension Rate):

Primer extension activity per unit was determined based on each of thecalculated specific activity values. The primer extension reaction wasperformed using a substrate (primed DNA) obtained by annealing a³²P-radiolabeled oligonucleotide to an M13 phage single-strand DNA (7kb). After 10 μL of a reaction mixture (5 nM M13 primed DNA, 0.2 mMdNTP, 50 mM Tris-HCl (pH 8.0), 1.5 mM MgCl₂, 50 mM KCl, 0.1%TritonX-100, and 100 mL, BSA) was reacted at 72° C. for 5 minutes, thereaction was terminated by adding 2.5 μL of 6×loading buffer (300 nMNaOH, 6 mM EDTA, 18% Ficol 400, 0.15% BCG, and 0.25% XC). The reactionproduct was separated by agarose gel electrophoresis (agarose gel wasprepared at a concentration of 1% in 50 mM NaOH and 1 mM EDTA) underalkaline conditions, and after the electrophoresis, the product wasdetected by autoradiography (using an image analyzer (FLA-5000,Fujifilm)).

The strand length of the reaction product was determined from theobtained image by comparing it with a size marker, whereby the strandlength of the synthetic product obtained per unit time (1 min) wascalculated. As a result, it was shown that whereas the wild-type Taqpolymerase had an extension rate of 3.89 kb/min, Taq E742A (refer toPatent Document 3: JP 4193997 given above) had an extension rate of 10.7kb/min, the mutant Exo⁻ whose 5′-3′ exonuclease activity residues weresubstituted in a site-specific manner had an extension rate of 9.17kb/min, and Exo⁻+E742A produced by crossing Taq B742A with Exo⁻ had arate of 15.4 kb/min.

3. Reverse Transcriptional Activity:

The intensity of the activity of incorporating deoxyribonucleotides on aRNA template strand was determined for each of the purified DNApolymerases. In order to perform the reaction, the DNA polymerase wasadded to a solution containing 20 ng/μL of poly(rA)*p(dT), 10 μM dTTP,440 nM [³H]-dTTP, 50 mM Tris-HCl (pH 8.0), 1 mM MnCl₂, 50 mM KCl, 0.1%TritonX-100, and 100 μg/mL of BSA, and the mixture was reacted at 60° C.for 10 minutes; then, 10 μL of the mixture was spotted onto DE81 paper.After air-dried, the paper was washed with an aqueous 5% disodiumhydrogenphosphate solution to remove unreacted nucleotides. The washingwas repeated three times each for 10 minutes. After the DE81 paper wasdried, radiation was measured by a liquid scintillation counter, wherebythe amount of [³H]-dTMP incorporated in the poly(rA)*p(dT) due to theDNA polymerase activity was calculated to determine enzyme activity (inunit). One unit is defined as the amount of enzyme, i.e., DNApolymerase, required to incorporate 10 nmol of nucleotides at 72° C. for30 minutes. The specific activity was calculated for each enzyme.

First, a Tth polymerase with strong reverse transcriptional activity wasprepared for use as a positive control. It was revealed that the Tthpolymerase purified by the present inventors showed a reversetranscriptional activity of 1.54×10 U/mg, while the wild-type Taq DNApolymerase which was also purified by the inventors showed a reversetranscriptional activity of 0.42×10⁴ U/mg (corresponding to a relativeactivity of 27% with respect to the Tth polymerase in the presentstudy).

The reverse transcriptional activity of each of the mutants constructedaccording to the present invention was determined using the sameconditions on the basis of its nucleotide incorporation activity. As aresult, it was shown that Taq R651E and Tth P653E showed a reversetranscriptional activity of 2.09×10⁴ U/mg and 2.54×10⁴ U/mg,respectively. These results mean that Taq R651E and Tth P653E showedactivities 1.36 and 1.65 times, respectively, greater than the wild-typeTth polymerase, whose reverse transcriptional activity is taken as 1.

The properties of the mutant Taq polymerases are summarized in thefollowing table.

TABLE 2 Purified Protein DNA-DNA RNA-DNA from 500 Specific RelativeSpecific Relative Extension Date DNA mL culture Activity activityActivity activity DNA RNA Polymerase (mg) (×10⁵ U/mg) (%) (×10³ U/mg)(%) (kb/U · min) (bp/pmol · min) Taq WT (SEQ ID NO: 12) 1.65 5.11 1004.24 27.5 3.89 14.0 Taq E742A 0.67 5.00 97.7 10.7 Taq A743H 0.89 3.8477.1 5.80 294L WT 0.48 6.67 131 0.11 294L E742A 0.56 12.7 249 0.29 303EWT 0.71 6.50 127 0.11 303E E742A 0.63 13.5 254 0.26 Exo · WT (SEQ ID NO:16) 0.54 3.79 74.2 9.17 Exo · E742A (SEQ ID NO: 18) 0.27 3.90 76.3 15.4Taq R651E (SEQ ID NO: 20) 1.12 3.44 67.3 20.9 136 4.36 45.4 Taq T664R0.65 2.03 39.8 7.74 Taq G568R 0.21 2.98 58.2 2.03 Taq S674R 0.31 4.5689.3 6.00 Tth WT (SEQ ID NO: 14) 0.68 3.66 71.5 15.4 100 14.6 117 TthP653E (SEQ ID NO: 22) 0.66 3.37 66.0 25.4 165 13.3 181

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1 PCR primer Taq117119A-F

SEQ ID NO: 2 PCR primer Taq117119A-R

SEQ ID) NO: 3 PCR primer Taq142144A-F

SEQ ID NO: 4 PCR primer Taq 142144A-R

SEQ ID NO: 5 PCR primer E742A-F

SEQ ID 3 NO: 6 PCR primer E742A-R

SEQ ID NO: 7 PCR primer R651E-F

SEQ ID NO: 8 PCR primer R651E-R

SEQ ID NO: 9 PCR primer P653E-F

SEQ ID NO: 10 PCR primer P653E-R

SEQ ID NO: 11 Taq WT nucleotide sequence

SEQ ID NO: 12 Taq WT amino acid sequence

SEQ ID NO: 13 Tth WT nucleotide sequence

SEQ ID NO: 14 Tth WT amino acid sequence

SEQ ID NO: 15 Taq Exo⁻ nucleotide sequence

SEQ ID) NO: 16 Taq Exo⁻ amino acid sequence

SEQ ID NO: 17 Taq Exo⁻+E742A nucleotide sequence

SEQ ID NO: 18 Taq Exo⁻+E742A amino acid sequence

SEQ ID NO: 19 Taq R651E nucleotide sequence

SEQ ID NO: 20 Taq R651E amino acid sequence

SEQ ID NO: 21 Tth P653E nucleotide sequence

SEQ ID NO: 22 Tth P653E amino acid sequence

1-25. (canceled)
 26. A DNA polymerase having a reverse transcriptionalactivity which is any one of (a) to (c) mentioned below: (a) a DNApolymerase comprising: an amino acid sequence modified from the aminoacid sequence of SEQ ID NO: 12, which has a substitution of the arginineresidue at position 651 by an amino acid residue having a negativelycharged side chain; or an amino acid sequence that is modified from anamino acid sequence of a Family A DNA polymerase derived from athermophilic eubacterium, which has a substitution of an amino acidresidue corresponding to position 651 in SEQ ID NO: 12 by an amino acidresidue having a negatively charged side chain; (b) a DNA polymerasecomprising an amino acid sequence modified from the amino acid sequenceof the DNA polymerase as recited in (a), which has a substitution,deletion, insertion and/or addition of one to nine amino acid residueswhich exclude the amino acid residue substituted in (a); and (c) a DNApolymerase comprising an amino acid sequence that is at least 95%identical to the amino acid sequence of the DNA polymerase as recited in(a), with the proviso that the amino acid residue substituted in (a) isthe same as the amino acid residue in (a).
 27. The DNA polymerase asrecited in claim 26, wherein the Family A DNA polymerase derived from athermophilic eubacterium is selected from the group consisting of DNApolymerases derived from Thermus aquaticus or DNA polymerases derivedfrom Thermus thermophilus.
 28. The DNA polymerase as recited in claim 26or 27, wherein the DNA polymerase of (a) is: (a1) a DNA polymerasecomprising an amino acid sequence modified from the amino acid sequenceof SEQ ID NO: 12, which has a substitution of the arginine residue atposition 651 by an amino acid residue having a negatively charged sidechain.
 29. The DNA polymerase as recited in claim 28, wherein the DNApolymerase of (a1) comprises the amino acid sequence of SEQ ID NO: 20.30. The DNA polymerase as recited in claim 28, wherein the DNApolymerase of (b) or (c) has a reverse transcriptional activity of atleast 16.0×103 U/mg.
 31. A polynucleotide which is any one of (A1) to(D1) mentioned below: (A1) a polynucleotide comprising the nucleotidesequence of SEQ ID NO: 19; (B1) a polynucleotide comprising a nucleotidesequence encoding the DNA polymerase as recited in claim 28; (C1) apolynucleotide that hybridizes under stringent conditions with apolynucleotide comprising a complementary sequence to the nucleotidesequence of SEQ ID NO: 19, and which encodes a DNA polymerase; and (D1)a polynucleotide encoding a DNA polymerase comprising a sequence atleast 95% identical to the nucleotide sequence of SEQ ID NO:
 19. 32. Thepolynucleotide as recited in claim 30, wherein the DNA polymerase in(C1) or (D1) has a reverse transcriptional activity of at least 16.0×103U/mg.
 33. The DNA polymerase as recited in claim 26 or 27, wherein theDNA polymerase of (a) is: (a2) a DNA polymerase comprising an amino acidsequence modified from the amino acid sequence of SEQ ID NO: 14, whichhas a substitution of the proline residue at position 653 by an aminoacid residue having a negatively charged side chain.
 34. The DNApolymerase as recited in claim 33, wherein the DNA polymerase of (a2)comprises the amino acid sequence of SEQ ID NO:
 22. 35. The DNApolymerase as recited in claim 33, wherein the DNA polymerase of (b) or(c) has a reverse transcriptional activity of at least 16.0×103 U/mg.36. A polynucleotide which is any one of (A2) to (D2) mentioned below:(A2) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:21; (B2) a polynucleotide comprising a nucleotide sequence encoding theDNA polymerase as recited in claim 33; (C2) a polynucleotide thathybridizes under stringent conditions with a polynucleotide comprising acomplementary sequence to the nucleotide sequence of SEQ ID NO: 21, andwhich encodes a DNA polymerase; and (D2) a polynucleotide encoding a DNApolymerase comprising a sequence at least 95% identical to thenucleotide sequence of SEQ ID NO:
 21. 37. The polynucleotide as recitedin claim 36, wherein the DNA polymerase in (C2) or (D2) has a reversetranscriptional activity of at least 16.0×103 U/mg.
 38. A DNA polymerasehaving a primer extension activity using DNA as a template which is anyone of (a4) to (c4) mentioned below: (a4) a DNA polymerases comprisingan amino acid sequence modified from the amino acid sequence of SEQ IDNO: 12, which has a substitution of at least one selected from theglutamic acid residue at position 117, the asparatic acid residue atposition 119, the asparatic acid residue at position 142, and theasparatic acid residue at position 144, as well as the glutamic acidresidue at position 742 by an amino acid residue having a non-polaraliphatic side chain; (b4) a DNA polymerase comprising an amino acidsequence modified from the amino acid sequence of the DNA polymerase asrecited in (a4), which has a substitution, deletion, insertion and/oraddition of one to nine amino acid residues, with the proviso that aminoacid residues corresponding to at least one selected from positions 117,119, 142 and 144, as well as position 742, in the amino acid sequence ofthe DNA polymerase as recited in (a4) remain the same; and (c4) a DNApolymerase comprising an amino acid sequence that is at least 95%identical to the amino acid sequence of the DNA polymerase as recited in(a4), with the proviso that amino acid residues corresponding to atleast one selected from positions 117, 119, 142 and 144, as well asposition 742, in the amino acid sequence of the DNA polymerase asrecited in (a4) remain the same.
 39. The DNA polymerase as recited inclaim 38, wherein the DNA polymerase of (a4) comprises the amino acidsequence of SEQ ID NO:
 18. 40. The DNA polymerase as recited in claim 38or 39, wherein the DNA polymerase of (b4) or (c4) has a primer extensionactivity using DNA as a template, of at least 4.00 kb/U·min.
 41. Apolynucleotide which is any one of (A4) to (D4) mentioned below: (A3) apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 17; (B4)a polynucleotide comprising a nucleotide sequence encoding the DNApolymerase as recited in claim 38; (C4) a polynucleotide that hybridizesunder stringent conditions with a polynucleotide comprising acomplementary sequence to the nucleotide sequence of SEQ ID NO: 17, andwhich encodes a DNA polymerase; and (D4) a polynucleotide encoding a DNApolymerase comprising a sequence at least 95% identical to thenucleotide sequence of SEQ ID NO:
 17. 42. The polynucleotide as recitedin claim 41, wherein the DNA polymerase of (C4) or (D4) has a primerextension activity using DNA as a template, of at least 4.00 kb/U·min.43. A recombinant vector comprising the polynucleotide as recited inclaim
 32. 44. A transformant comprising the recombinant vector asrecited in claim
 43. 45. A process for preparing the DNA polymerase asrecited in any one of claims 1 to 5, 8 to 10, and 18 to 20, the processcomprising a step of culturing the transformant as recited in claim 24.